The present studies determine in greater detail the molecular mechanisms upstream of the CD95
death receptor by which
geldanamycin heat shock protein 90 inhibitors and
mitogen-activated protein kinase/
extracellular signal-regulated kinase kinase 1/2 (MEK1/2) inhibitors interact to kill
carcinoma cells. MEK1/2 inhibition enhanced
17-allylamino-17-demethoxygeldanamycin (
17AAG) toxicity that was suppressed in cells deleted for mutant active RAS that were nontumorigenic but was magnified in isogenic tumorigenic cells expressing Harvey RAS V12 or Kirsten RAS D13. MEK1/2 inhibitor and
17AAG treatment increased intracellular Ca(2+) levels and reduced
GRP78/BiP expression in a Ca(2+)-dependent manner.
GRP78/BiP overexpression, however, also suppressed drug-induced intracellular Ca(2+) levels. MEK1/2 inhibitor and
17AAG treatment increased
reactive oxygen species (ROS) levels that were blocked by quenching Ca(2+) or overexpression of
GRP78/BiP. MEK1/2 inhibitor and
17AAG treatment activated CD95 and inhibition of
ceramide synthesis; ROS or Ca(2+) quenching blocked CD95 activation. In SW620 cells that are patient matched to SW480 cells, MEK1/2 inhibitor and
17AAG toxicity was significantly reduced, which correlated with a lack of CD95 activation and lower expression of
ceramide synthase 6 (LASS6). Overexpression of LASS6 in SW620 cells enhanced drug-induced CD95 activation and enhanced
tumor cell killing. Inhibition of
ceramide signaling abolished drug-induced ROS generation but not drug-induced cytosolic Ca(2+) levels. Thus, treatment of
tumor cells with MEK1/2 inhibitor and
17AAG induces cytosolic Ca(2+) and loss of
GRP78/BiP function, leading to de novo
ceramide synthesis pathway activation that plays a key role in ROS generation and CD95 activation.